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Detection and partial characterization of a UV-damaged-DNA binding activity highly expressed in zebrafish (Danio rerio) embryos

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Abstract

The expression of distorted DNA-binding factors was studied in developing zebrafish (Danio rerio) using UV-damaged DNA as the binding target. A strong and high-shifting binding activity was detected in the extracts of zebrafish early embryos (12 h after fertilization), and the expression of this activity dramatically decreased in 60 to 84-h-old zebrafish. The embryonic extracts produced a similar pattern of high-shifting complexes after incubating with a CPD-specific or a 6-4PP-specific probe, while different types of low-shifting complexes were generated by the extracts of 84-h-old larvae. The formation of high-shifting complexes was suppressed in the presence of NaCl at 0.25 M or higher concentrations, yet the production of low-shifting complexes was stimulated by increasing salt concentration. The binding activity expressed in zebrafish embryos was apparently unrelated to NER-associated damage-recognition protein XPA, since two polypeptides recognized by an anti-human XPA antibody were detected only in 84-h-old zebrafish extracts. A competitive binding assay indicated that both CPDs and 6-4PPs were recognized by the same binding activity expressed in 12-h-old zebrafish, and this activity contained at least two protein fractions that were eluted from a DEAE-cellulose column by NaCl at 0.1 M and 0.2 M. UV crosslinking of the two NaCl eluates to a 6-4PP probe produced covalent complexes with the same electrophoretic mobility except one 34-kDa complex generated by the 0.1 M NaCl eluate, suggesting the existence of two multisubunit damage-recognition protein complexes in zebrafish embryos. UV-binding factors found in 12-h-old zebrafish embryos may be involved in processing developmental stage-specific DNA structures similar to UV-damaged DNA.

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Authors and Affiliations

  1. Institute of Marine Biotechnology, National Taiwan Ocean University, Keelung, 20224, Taiwan, Republic of China

    T. Hsu, C.S. Cheng, C.Y. Shih & F.L. Yeh

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  1. T. Hsu
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  2. C.S. Cheng
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  3. C.Y. Shih
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  4. F.L. Yeh
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Hsu, T., Cheng, C., Shih, C. et al. Detection and partial characterization of a UV-damaged-DNA binding activity highly expressed in zebrafish (Danio rerio) embryos. Fish Physiology and Biochemistry 25, 41–51 (2001). https://doi.org/10.1023/A:1019755900858

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